Tandem time of flight mass spectrometer and method of use

ABSTRACT

To provide comprehensive (i.e. rapid and sensitive) MS-MS analysis, the inventor employs a time-nested separation, using two time-of-flight (TOF) mass spectrometers. Parent ions are separated in a slow and long TOF 1 , operating at low ion energy (1 to 100 eV), and fragment ions are mass analyzed in a fast and short TOF 2 , operating at much higher keV energy. Low energy fragmentation cell between TOF 1  and TOF 2  is tailored to accelerate fragmentation and dampening steps, mostly by shortening the cell and employing higher gas pressure. Since separation in TOF 1  takes milliseconds and mass analysis in TOF 2 —microseconds, the invention provides comprehensive MS-MS analysis of multiple precursor ions per single ion pulse. Slow separation in TOF 1  becomes possible with an introduction of novel TOF 1  analyzers. The TOF-TOF could be implemented using a static TOF 1 , here described on the examples of spiratron, planar and cylindrical multi-pass separators with griddles spatial focusing ion mirrors. Higher performance is expected with the use of novel hybrid TOF 1  analyzers, combining radio frequency (RF) and quadratic DC fields. RF field retains low-energy ions within TOF 1  analyzer, while quadratic DC field improves resolution by compensate for large relative energy spread.

FIELD OF THE INVENTION

The invention relates to the area of mass spectrometry, and more inparticularly is concerned with a method of high-throughput,comprehensive tandem mass spectrometry in apparatus, including twotime-of-flight mass spectrometers.

BACKGROUND OF THE INVENTION

Mass spectrometers are devices which vaporize and ionize a sample andthen use static or dynamic electric fields to measure the mass-to-chargeratios of the ions formed. Tandem mass spectrometry is used forstructural analysis and the identification of compounds in complexmixtures. In every application the MS-MS procedure has the same sequenceof operations:

-   -   Mass selection of parent ions of a single mass-to-charge ratio        (m/z);    -   Fragmentation of those ions;    -   Mass analysis of the fragments.        Though there is a large variety of tandem MS-MS instruments with        their own strength and weakness, all of them have one common        feature—all of them use one parent ion at a time. The rest of        ion species are removed out of the primary ion beam and lost.

Triple quadrupole instrument is the most common MS-MS instrument.Continuous ion source, like e.g. electrospray (ESI), introduces ionsinto a first quadrupole mass filter, which is tuned, such that onlyions-of-interest pass the mass filter. The rest of primary beamcomponents are rejected and lost. Selected ions are transmitted into aso-called ‘collision induced dissociation’ (CID) cell, filled with gasat mTorr pressure and equipped with a radio frequency (RF) quadrupoleguide. The kinetic energy of injected ions is controlled byelectrostatic bias of mass filter and it is adjusted to induce ionfragmentation via gas collisions. Fragment ions are collisional dampenedin CID cell and then introduced into a second quadrupole for massanalysis. Since mass scanning in a second quadrupole takes time andcauses additional ion losses by factor of c.a. 1000, triple quadrupoleinstruments are mostly used for detection of known species with knownmasses of parent and fragment ions.

Introduction of quadrupole-time-of-flight tandem mass spectrometers(Q-TOF) strongly enhanced throughput of MS-MS instruments (see Morriset.al. Rapid Commun. Mass Spectrom. v.10, pp.889-896, 1996). The triplequadrupole was modified, such that second quadrupole mass filter wasreplaced by an orthogonal TOF MS (oa-TOFMS). This substitution gave anadvantage of parallel analysis of all fragment at once and, hence,higher sensitivity and faster acquisition in a second MS, as well asenhanced resolution and mass accuracy of second MS. However, quadrupoleis still used for parent ion selection, accompanied by rejection of allion species but one. The idea of parallel analysis has not been extendedonto parent ions.

Another common MS-MS device uses Paul ion trap mass spectrometer (ITMS),well described in March, R. E., Hughes R, J. Quadrupole storage massspectrometry, Willey-Interscience, New York 1989. Ions, produced in theion source, are periodically injected into an ITMS and are trappedwithin the ITMS by radio-frequency (RF) field. ‘Unwanted’ species areremoved by e.g. applying a broadband resonant AC signal, so that onlyions-of-interest remain in the trap. Selected parent ions are thenexcited by a separate AC field, resonant with the secular motion of theprecursor. Parent ions gain kinetic energy and fragment in energeticcollisions with a buffer gas. Fragments are mass analyzed using aresonant ejection technique. The amplitude of RF field is ramped suchthat ions leave the trap sequentially according to their m/z values.

It also has been known to couple 3-D Paul trap with a TOF analyzer formore accurate mass analysis of fragment ions, see Quin and D. Lubman,Rap. Commun. Mass. Spectrom. 10, 1079, 1996 and WO 099/39368 byShimadzu. Linear ion trap (LIT) has been coupled to TOF analyzer in U.S.Pat. No. 5,847,386 by D. Douglas, in U.S. Pat. No. 6,111,250 by Sciex;in U.S. Pat. No. 6,020,586 by Analytica and in WO 01/15201 by U of NewHampshire. All ion trap tandems are mostly oriented on multiple stageMS-MS analysis. Parent ions are selected with a loss of other ioncomponents.

Recently introduced tandem time-of-flight mass spectrometers (TOF-TOF)are the closest prototypes to the below described invention bysimilarity of employed hardware. Examples of TOF-TOF are described inU.S. Pat. No. 5,032,722 by Schlag et.al., U.S. Pat. No. 5,464,985 by T.J. Kornish et.al., U.S. Pat. No. 5,854,485 by T. Bergmann, U.S. Pat. No.______ WO99/40610 by M. L. Vestal and in WO99/01889 by C. Hop. In allTOF-TOF tandems, a pulsed ion beam is time separated in a first,high-energy TOF and filtered by timed ion selector, so that onlyions-of-interest pass into CID cell. The CID cell is filled with gas ata low gas pressure (usually below 1 mtorr) to introduce nearly singlehigh energy collision with buffer gas, sufficient for ion fragmentation,but still retaining short duration of ion packet. A pulsed beam offragment ions is analyzed in a second, high energy TOF. To handle largeenergy spread of fragment ions, second TOF employs either quadraticfield potential or an additional pulsed acceleration.

In WO 00/77823 by A. Verentcfikov, a variation of TOF-TOF tandem employsslow injection of parent ions into a CID cell with collisional dampeningof fragments and subsequent injection into an orthogonal TOF. Theinstrument is the closest prototype of the invention, consideringemployed components. Collisional dampening in the fragmentation cellimproves ion beam characteristics in-front of the second TOF and allowshigh resolution and accurate measurements of fragment ion masses. Thefirst TOF operates at 1 kV energy and a short time scale. Time gate infront of CID cell admits only one parent ion mass at a time.

In all described tandems the first mass analyzer (either quadrupole, iontrap or TOF) selects one parent mass in a time and rejects all othercomponents. In some applications, like drug metabolism studies, it isacceptable to follow a single compound of interest. In the case ofcomplex mixtures (like protein characterization out of gels), however,it is necessary to analyze multiple parent ions. Using existingtechniques, sequential MS-MS analysis of multiple precursors is tediousand insensitive.

Recently introduced tandem IMS-CID-TOF, employs principle of time-nestedacquisition, potentially to be implemented without ion losses, WO00/70335 by D. Clemmer. Since separation in ion mobility spectrometer(IMS) occurs in millisecond time scale and TOF mass spectrometry—inmicrosecond scale, it become possible to acquire fragment spectra foreach ion mobility fraction. The disadvantage of the technique is a poorIMS separation with mobility resolution below R=50, which corresponds tomass resolution of about 10. Since, IMS-TOF tandem employs a principleof comprehensive tandem mass spectrometry with time-nested acquisition,it is selected as a prototype of the invention.

The idea of MS-MS analysis without parent ion losses is also used in WO01/15201 by B. Reinhold and A. Verentchikov. Ions are selected byresonant excitation and moved between ion traps without rejecting otherionic components. The procedure is tedious and takes long time, whileions coming from the ion source are lost. So-called parallel ionprocessing is employed in multiple ion traps in WO92/14259 by Kirchner,where the beam is split between multiple traps. Time is saved by loosingsensitivity.

There is still a need for an instrument providing rapid and sensitiveMS-MS analysis for multiple parent ions in parallel without rejectingions coming out of ion source. Such instrument would further improve athroughput of MS-MS analysis, desirable in analysis of complex mixtures.

SUMMARY OF THE INVENTION

The present inventor has realized, that one can implement the principleof nested time separation using two time-of-flight (TOF) massspectrometers—slow TOF1 for parent ion separation and fast TOF2 forfragment mass analysis. Thus, general method of tandem mass spectrometryof the invention employs two time of flight separations, wherein for thesame mass-to-charge ratio, flight time in the first separation step ismuch longer than flight time in the second separation step andmultiplicity of parent ions are separated, fragmented and mass analyzedper single ion injection out of ion source.

Tandem mass spectrometer of the invention comprises a pulsed ion source,a time-of-flight mass spectrometer (TOF1) for time separation of parentions, a fragmentation cell, a second time-of-flight mass spectrometer(TOF2) for mass analysis of fragment ions and a data acquisition system.Contrary to prototype TOF-TOF systems, flight time in the TOF1 issubstantially larger than both passage time through fragmentation celland flight time in the TOF2. Prolonged separation in TOF1, typically inmillisecond range, could be achieved by operating longer TOF1 at muchlower kinetic energy, typically around 1 to 100 eV, while using shorterTOF2 at 3 to 10 keV energy. Time between arrival of adjacent parent ionspecies becomes sufficient to fragment and mass analyze fragments. Thus,the invention allows rapid MS-MS analysis of multiple parent ions inreal time without rejecting parent ions. The MS-MS acquisition cyclelasts few milliseconds and can be repeated multiple times to improvesensitivity and signal quality.

To avoid ion losses the ion source is operated in a pulsed mode at about100 Hz repetition rate, compatible with millisecond time of MS-MS cycle.Matrix Assisted Laser Desorption/Ionization (MALDI) ion source is oneexample of usable pulsed ion source. The invention is also compatiblewith a wide variety of continuous ion sources, like ESI, MALDI with gascooling, Chemical Ionization and gas filled Photo-ionization ionsources. Ion flow is continuously accumulated within storage radiofrequency (RF) device and is periodically pulse ejected into the TOF1.The said storage device can be either Paul trap or storage multipole,preferably quadrupole.

To the best knowledge of the author, the novel time-nested TOF-TOFmethod can not be implemented on existing TOF-TOF instruments withoutsevere sacrifice of performance. The invention discloses five novel TOF1separators, operating at low ion energy (1 to 100 eV) to expandseparation time.

Two of those novel TOF1 analyzers employ combination of confining radiofrequency (RF) field with DC quadratic field, providing temporalfocusing of ion beam with a large relative energy spread. Thoseanalyzers are capable of operating at particularly low ion energy from 1to 10 eV. In one preferred embodiment, the novel TOF1 analyzer comprisesa linear multipole ion guide, preferably quadrupole, surrounded by DCmirrors. DC mirrors on both ends are turned on and off to provide ioninjection from one TOF1 end, multiple ion reflections and subsequent ionrelease from another end. In another preferred embodiment, the novelTOF1 analyzer comprises two external rows of DC electrodes and twointernal rows of RF-only rods, oriented across TOF1 axis. The structureforms two dimensional RF-tunnel combined with quadratic potentialdistribution along the TOF axis. Ions are injected into the TOF1 atsmall angle to the axis, experience multiple reflections along the axis,slowly shift across the axis and leave TOF1 after several reflections.

Another three novel analyzers are electrostatic devices, operating atmedium energy around 100 eV. One of them, ‘spiratron’ comprises a pairof coaxial cylindrical electrodes with DC voltage applied between them.Ions are injected between said electrodes at small angle to their axis.Medium energy (100 eV) ions turn around central electrode while driftingslowly along the axis. After a number of turns ions leave TOF1 through acut-off boundary, which is formed by double sided printed circuit boardto avoid DC field disturbance. Other two electrostatic separators areplanar and cylindrical multi-pass analyzers, employing griddles mirrors,simultaneously acting like a lens. Effective flight path is extended byuse of a multi-pass mode, so that 10 ms time scale is achieved in-spiteof a higher energy (compared to RF assisted TOF1).

The invention is compatible with variety of fragmentation methods—in gascollisions, in collisions with surface and by light. The design offragmentation cells is trimmed to reduce transmission time and timespread. CID cell is chosen short (around 1 cm), filled with gas atrelatively high pressure (above 0.1 mbar) and supplemented by axial DCfield to accelerate transmission and to modulate ion beam synchronouswith TOF2. The surface induced dissociation (SID) cell uses pulsed lensto provide spatial focusing together with temporal focusing (bunching).Ions are ejected out of SID cell by pulsing probe potential,synchronized (though with time shift) with bunching lens and TOF2pulses.

Though, choice of second time-of-flight analyzer is not critical, theTOF with orthogonal ion injection (o-TOF) is more suitable in majorityof tandem examples. In order to improve efficiency of orthogonalinjection (so-called duty cycle), it is preferred to eject ions out offragmentation cell synchronous and slightly prior to orthogonalinjection pulses.

The TOF-TOF tandem is expected to separate parent ions at moderateresolution, mostly limited by speed of second TOF MS, e.g. 10 us. Theestimated resolution of TOF1 in the order of 300 (see detaileddescription) is still sufficient to isolate a group of isotopes ofparent ions and is much higher than resolution of parent separation inion mobility spectrometer—a prototype of the invention. Higherresolution of separation could be achieved in longer TOF1, or byperiodic selection of ions by time gate in front of CID cell.

The invention allows multiple strategies of data acquisition. In asimplest and robust approach, MS-MS data are acquired continuously andMS-MS spectra of multiple parent ions are reconstructed afterwards. Itis wiser, though, to perform MS-MS analysis in two stages. At first,MS-only stage, parent ions are continuously admitted into TOF2 for massanalysis of parent ions. Information on masses of parent ions is usedfor a second MS-MS stage. Time gate opens only at time of arrival ofmultiple parents of interest to improve resolution of parent ionseparation and to avoid signal from chemical background. TOF2 signal isalso acquired for selected time windows only to reject meaningless dataflow. Similar information on parent ions may be obtained using anoptional on-line detector located anywhere after TOF1.

In addition to highly sensitive and rapid MS-MS analysis the inventionprovides multiple types of MS-only analysis. TOF1 alone can be used forMS only analysis for a sake of spreading peaks in time, avoidingdetector saturation and using inexpensive and slow transient recorder.Better quality spectrum of parent ions could be acquired in TOF2 whileusing TOF1 in a pass mode. So-called “Parent scan”, i.e. spectrum ofparent ions having a set of specific fragments, can be reconstructedfrom MS-MS data, averaged in multiple source injections. The data couldbe finally stored for parents masses only.

Since MS/MS spectra are acquired for all precursor ions of interest in asingle ion injection, the invention provides an exceptional speed ofMS/MS analysis, estimated as 10 to 30 full cycles a second. The speed ofMS-MS analysis is compatible with time scale of chromatographicseparation, thus, a real time LC-MS-MS analysis is possible without anyprior limitations, such as “data dependent acquisition”, currentlyemployed in ion traps and Q-TOF. High acquisition speed and sensitivityof the invented MS-MS tandem also opens an opportunity for using nestedLC-LC analysis up-front.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood referring to the following description taken in conjunctionwith the accompanying drawings in which:

FIG. 1 is a block diagram, illustrating the method of the invention.

FIG. 2 is a timing diagram of operation of tandem TOF-TOF massspectrometer.

FIG. 3 is a schematic of novel in-line TOF1.

FIG. 4 is a schematic of novel W-shape TOF1.

FIG. 5 is a schematics of vacuum pulsed MALDI ion source.

FIG. 6 is a schematic of pulsed MALDI ion source with collisionaldampening.

FIG. 7 is a schematics of continuous ion source with pulsing storagequadrupole.

FIG. 8 is a schematic of CID cell.

FIG. 9 is a schematic of SID cell.

FIG. 10 is a schematic of orthogonal TOF2.

FIG. 11 is a schematic of coaxial TOF2

FIG. 12 is a schematics of TOF-TOF with in-line TOF1 and CID cell.

FIG. 13 is a schematics of TOF-TOF with W-shape TOF1 and SID cell.

FIG. 14 is a schematic of TOF-TOF with static coaxial TOF1.

FIG. 15 is a schematic of planar electrostatic multi-pass TOF1.

FIG. 16 is a schematics of cylindrical electrostatic multi-pass TOF1.

DETAILED DESCRIPTION OF THE INVENTION

Method

A method of tandem mass spectrometry analysis of the invention comprisesthe steps of:

-   -   1. generating an ion pulse in an ion source, containing a        mixture of different analyte ions;    -   2. separating analyte ions in time within a first time-of-flight        mass spectrometer, operating at low energy, and, thus,        generating a train of ion packets in a sequence of their masses,    -   3. sequentially fragmenting analyte ions without mixing said        separated ion packets;    -   4. rapidly mass analyzing fragment ions within a second        time-of-flight mass spectrometer at a time scale much shorter,        than time scale of the first separation step;    -   5. acquiring fragment mass spectra for multiple analyte ion        mass-to-charge ratios at a single ion pulse out of the ion        source, and    -   6. optionally, summing the fragment spectra for each analyte        ions over multiple source pulses.    -   7. The key of the method is arranging separation time in the        first TOF much longer than fragmentation time and time of        fragment mass analysis for the same mass-to-charge ratio.        Substantial difference in time scales is utilized to separate,        fragment and mass-analyze fragments for multiplicity of parent        ions per single ion injection out of the ion source. Substantial        difference in time scale is achieved by selecting longer flight        path and/or lower ion energy in the first TOF.

Block Diagram

Referring to FIG. 1, the method is illustrated by a block diagram ofmajor tandem MS-MS components. The generic TOF-TOF instrument withtime-nested acquisition (11) comprises sequentially communicating pulsedion source (12), a first time-of-flight mass spectrometer—TOF1 (13), afragmentation cell—CID/SID (14), a second time-of-flight massspectrometer TOF2 (15) and a data system (16) for time-nestedacquisition. The pulsed ion source is biased compared to the TOF1spectrometer at a small potential difference by voltage supply (17), andthe TOF1 is biased compared to the CID cell at potential difference byvoltage supply (18). An optional timed gate (19) may be inserted betweenthe TOF1 (13) and the CID cell (14) to enhance TOF1 separation.

Operation

Briefly, in operation, the pulsed ion source generates an ion pulse ofanalyte (parent) ions and injects ions into the TOF1 at a small energy,between 1 to 10 eV, controlled by a voltage supply (17). This is the keydifference between the current invention and a prior art, since TOFspectrometers are usually operated at energies between 3 and 30 keV.Separation in TOF1 occurs in several milliseconds. As a guiding examplelet us consider effective length of TOF1=8 m, ions energy E=3 eV and ionmass m=1000 a.u. In such example ion velocity is V=800 m/s and flighttime is 10 ms. Time separated parent ions are sequentially ejected outof TOF1 into the CID cell at an increased energy, controlled by DC biasbetween TOF1 and the cell. Energetic collisions with gas moleculesconvert parent ions into fragments. Subsequent gas collisions causecollisional dampening of fragment ions. Fragments rapidly travel throughthe cell and are injected into the TOF2 spectrometer. TOF2 separatesfragment ions at a much shorter time scale, between 10 and 100 us.Drastic difference in time scales of TOF1 and TOF2 allows dataacquisition of multiple fragment spectra, corresponding to differentparent ions between source pulses. The specialized data acquisitionsystem (16) acquires multiple fragment spectra in a time-nested fashion,where individual spectra are not mixed together. Fragment spectra foreach parent ion are integrated over a number of ion source pulses. Thus,ion pulse, generated in the ion source, is used for acquiring a full setof MS-MS data for multiple parents without rejecting ions at all stages.

Time Diagram

Referring to FIG. 2, a typical time diagram illustrates the method ofthe invention, synchronization of individual devices and a principle oftime-nested data acquisition. The top graph (21) presents an acquisitioncycle, where ion injections occur every 10 ms, i.e. 100 times a second.Parent ions are separated in the TOF1 within 10 ms time, and the CIDcell receives a train of ion packets, aligned in accordance with parention mass, graph (22). Parent ions are partially fragmented in the cell,and because of a short transmission time in the cell, fragments arriveto the TOF2 almost simultaneously with their parents, graph (23). Eachnew family of ions (i.e. parents and daughters) is orthogonal pulsedinto the high energy TOF2 every 10 us, producing TOF2 spectra for eachparent mass—graph (24). Each TOF2 spectrum obtains a time tag of TOF2pulse relative to source pulse, i.e. TOF1 time tag. The spectra with thesame TOF1 time tag are, summed over multiple ion source pulses, as shownby dashed lines, connecting two TOF2 spectra with the same TOF1 timetag.

Robust Mode

In the above described operation mode, the time-nested acquisition isdone in a straightforward way. Instrument operation parameters remainthe same, regardless of the ion beam composition out of the ion source,and data are acquired all the time. All the information, like parent ionspectra and fragment spectra for various parents, is extracted in asubsequent data analysis.

Data Dependent Acquisition—DDA

In another operation mode, which should be called ‘data dependentacquisition’, MS-MS analysis occurs in two steps. On the first step,mass spectrum of parents is acquired in a TOF2, while TOF1 and CID cellpass ions continuously without fragmentation. On the second step, theinstrument is operated as MS-MS, i.e. the TOF1 separates parent ions,the fragmentation cell forms fragments, the TOF2 acquire fragment massspectra in the time-nested data fashion. The time-nested acquisition isenhanced by utilizing the information on parent ion masses and avoidingdata acquisition at blank times, when no parents are coming. An optionaltimed gate (19) may be used to enhance TOF1 separation as well assuppression of chemical noise. It is naturally expected, that ionpackets coming out of TOF1 are shorter, than the same ion packet at theexit of the CID cell. The timed gate admits ions only at multiple narrowtime windows, corresponding to arrival of parent ions. Such gatingsuppresses ion signal coming from chemical background and improvesdetection limit. Gate operation may also be used to enhance separationof pair of parent ions of close mass by sacrificing sensitivity. Severalsets of MS-Ms data are acquired, while timed gate admits only one parentmass of a pair in a time.

Having described general method, and for the purpose of clarity, thedetailed embodiments will be first discussed on the level of individualcomponents and only then presented as examples of integrated TOF-TOFapparatus. Though, some employed components are well known in the art,their configuration and parameters are altered to suit purposes of theinvention. To understand selected compromises, let us first look atmajor challenges in TOF-TOF method and apparatus.

General Objection

The method of the invention is highly counterintuitive, since it wouldbe referred as undoable for multiple reasons. One skillful in the artwould object that:

-   -   1. TOF1 resolution would be extremely low; since ion energy        spread in the source is comparable to ion energy in the TOF1;    -   2. TOF1 resolution would also suffer because of a large turn        around time (time spread, caused by initial velocity spread) in        a weak accelerating field;    -   3. Ion losses through the TOF1 are expected to be devastating,        because of expected large length of TOF1, and because of high        divergence of slow ion beam in the TOF1;    -   4. Ion losses are expected to be even higher, since vacuum stage        of TOF1 and gas filled CID cell should be separated by a small        aperture;    -   5. It also looks unlikely to have quick transmission through the        CID cell in the time scale of 10 to 100 us. Most existing CID        cells have 200 to 10,000 us time spread;    -   6. None of commercial data acquisition system, currently        employed in TOF technology, is capable of handling expected data        flow rate.

The above objections are mostly concentrated around TOF1 and arise fromknowledge on existing TOF mass spectrometers, operating at high energy.The inventor has realized that multiple schemes of TOF1 are capable ofslow separation with moderate resolution. Improvement of TOF1 resolutionis made by employing an ion mirror with quadratic potentialdistribution, known to compensate for energy spread. The phenomenon issimilar to elastic oscillations, where period does not depend onoscillation amplitude. Quadratic fields are well explored in TOF art.See Makarov et.al. in Int J. of Mass Spectrom and Ion Processes,v.146/147, 1995, pp.165-182. Unfortunately, such analyzers alsointroduce a large beam divergence. The inventor also realized that lowenergy TOF could be improved by introducing a radio frequencyconfinement of ion beam in at-least one direction. RF confinementeliminates ion beam divergence and also eliminates surface charging,crucial for low energy apparatuses. A novel type of TOF has been found,combining RF confinement with axial DC quadratic potential.

In-line TOF

Referring to FIG. 3, the preferred embodiment of novel low energytime-of-flight separator (31) comprises an RF-only multipole (32), twoelectrostatic mirrors (33) and pulse generators (34). Mirrors areconstructed of multiple electrodes, interconnected with a chain ofdividing resistors (35). External electrodes of mirrors (33) areconnected to pulse generators (34) and middle electrode of mirrors (33)being ground. End field is terminated by apertures (36), with potentialadjusted as a portion of full potential on pulse generators (34).

In operation, RF field provides radial confinement, shown by arrows (37)on FIG. 3. Radial RF confinement does not affect ion motion along theaxis. Axial parabolic electric field is formed by field penetrationbetween multipole rods. Parabolic field provides ion axial reflectionswith period, grossly independent on ion energy and proportional tosquare root of ion m/z. Pulsing potentials on mirror ends allowsswitching between ion injection into TOF1, ion reflections (39) withinTOF1 and subsequent ion release on the other end of TOF1. The effectiveflight path LEFF is N□+1 times higher than TOF1 length L, where N is anumber of full turns. Overall, RF confinement and multiple reflectionsallow prolonged time separation without ion losses, while quadraticpotential enhances TOF1 resolution and allows separation of a slow ionbeam with a high relative energy spread.

The ideal quadratic scheme is altered by presence of free flight segmenton the way in and the way out of TOF1. According to above citedpublication by Makarov et.al, even in case of substantial field freeflight, here c.a. 30% of LEFF, a mass resolution of 2000 is achievablefor ion pulses with relative energy spread up to 50%. To keep freeflight path below 0.3 LEFF, the scheme requires at least 5 reflections,corresponding to 2 full turns. It helps to increase Leff to 7.3 L butreduces mass range of parent ions to a factor of two, i.e. MMAX/MMIN<2.

W-TOF

Referring to FIG. 4, another preferred embodiment of novel low energytime-of-flight separator (41) comprises an RF channel (42), surroundedby a set of electrostatic electrodes (43), terminating electrodes (44),and a deflector (45). The RF channel is formed by multiple rods (46)with alternating RF phase and aligned along Y axis. Electrodes ofelectrostatic mirrors (43), are also aligned along Y axis, and areconnected via a chain of dividing resistors (47).

In operation, rods (46) with alternating RF potential form an RF tunnel,confining ions in Z direction. Potential on electrodes (43, 44) isdistributed by resistor chain to form quadratic potential along X axiswith minimum at the center plane of TOF. Field of external DC electrodespenetrates into the RF channel, providing a weaker but still quadraticpotential distribution. Not accounting fringing fields there is no fieldin Y direction. Ions are injected at a small angle to X axis and aredeflected by deflection plates (45) to double deflection angle for ionswith mean energy. The deflection reduces Y-spatial spread, caused byX-energy spread. Ion motion is combined of a slow drift along Ydirection and of multiple reflections along X direction. Overall, iontrajectories have a wave shape, ending at the boundary of the RF tunnel.Ions gain some spatial spread at the exit of TOF, which partiallycompensated by ion post-acceleration and focusing by a lens.

According to SIMION simulations by inventor, even at 50% energy spreadthe 50×30 cm device allows N=4 to 5 pairs of reflections without mixingions with adjacent turns. The effective flight path of the device equalsto L*□*N, and reaches LEFF=7.5 m. The RF field does not limit TOF1resolution up to R=1000. Obviously a second type of TOF1, which may becalled RF confined W-shape TOF, provides a simpler operation and longerflight path in TOF1, thus improving separation in TOF1, mostly limitedby ratio of flight time between two TOF analyzers. The complexity ofTOF1 could be reduced by using printed circuit board (PCB) assembly.

Answering Objections

In both novel TOF mass separators, the period of each reflection isgrossly independent on ion energy and is proportional to square root ofion m/z. Ions are confined by RF field and ion losses practicallyeliminated. Introduction of novel low-energy TOF analyzers makes thepresent invention practical, resolving the above mentioned objections:

-   -   1. High relative energy spread is compensated by quadratic        distribution of potential in the ion mirror, created by DC        electric field penetration into multipole guide or tunnel;    -   2. Because of TOF1 ability to operate at high relative energy        spread, it can operate at much lower ion energy and at a much        longer time scale, compared to conventional TOF. As a result,        the apparatus tolerates a much longer ion pulse out of the ion        source, and turn around time is no longer an obstacle;    -   3. Drastic difference in time scales of TOF1 and TOF2 allows        time-nested data acquisition;    -   4. Ion losses are practically avoided by guiding ions within        radio-frequency guide or tunnel;    -   5. Ion confinement by RF field and ion post-acceleration        in-front of the CID cell allow full transmission of ion beam        into the CID cell;    -   6. Time spread in the CID cell is reduced by using a short, high        pressure cell with an additional axial DC field, and    -   7. Transient recorder with a large and fast averaging memory is        recently introduced by Switzerland company Acquiris        (www.acquiris.com).

The detailed description continues on the level of individualcomponents: pulsed ion sources, fragmentation cell and TOF2,specifically tailored for purposes of the method and apparatus of theinvention. Particular attention will be paid to the issue of timespread.

Vacuum MALDI Source

Referring to FIG. 5, TOF-TOF method and apparatus of the inventionemploy a pulsed MALDI ions source (51), comprising a source housing(52), a sample plate (53) with analyzed sample (54), a pulsed laser(55), a low voltage power supply (54), and an exit aperture (56).

In operation,, samples for analysis are prepared within matrices knownin the art, and deposited on the sample plate (53). Pulsed laser (55)illuminates the sample and generates a short pulse of analyte ions. Ionsare known to be ejected with 300 to 600 m/s velocity, which correspondsto initial ion energy between 0.5 and 1.5 eV for 1 kD. ion. Ions areaccelerated by few Votls potential bias. One can estimate, that 1 kDions leave ion source with few microseconds time spread and less than 1eV energy spread. The major drawback of vacuum MALDI ion source is iontemporal instability, well described in conventional, high energy MALDI.The invention is likely to be applicable to softer MALDI ion sources,employing soft matrices or an infra-red laser. Temporal stability ofions is improved by collisional cooling, described below.

[Gas Filled Pulsed MALDI]

Referring to FIG. 6, TOF-TOF method and apparatus of the inventionemploys gas-filled pulsed MALDI ion source (61). The source (61)comprises features of vacuum MALDI source, such as a source housing(62), a sample plate (63) with analyzed sample (64), a pulsed laser(65), a low voltage power supply (66), and an aperture (67A); The source(61) also comprises a gas inlet (68), feeding gas into the housing (62),an additional pumping stage (69), terminated by exit aperture (67B) toreduce a gas load on TOF1 pump.

In operation, the source housing (62) is filled with buffer gas via thegas inlet (67). Gas pressure in the source housing is sustained between0.01 to 1 Torr to provide ion collisional cooling (see Verentchikov etal, ASMS Conference 1999 in www.asms.org). Differential pumping systemwith two 1 mm apertures (67A, B) and two conventional 2501/s turbo pumps(one pumping TOF1), sustains vacuum in TOF1 better than 1 E-6 torr. Thelaser pulse generates a rapid (1 to 3 ns) ion ejection out of sample.The laser (65) is a high-energy laser to enhance ion production.Collisions with buffer gas relax ion internal energy. Collisions withgas also dampen ion kinetic energy to nearly thermal energy −0.01 to 0.1eV, still retaining pulse property of ion beam. Ions are sampled by gasflow through the aperture, assisted by c.a 1V DC bias on the sampleplate. Ions are then accelerated to kinetic energy, controlled by DCbias between apertures (67A, B), and leave the ion source. Internallycold ions are stable and survive long separation in TOF1 without iondecomposition. Overall, gas dampening in the MALDI source benefitsTOF-TOF method of present invention, while leaving time and energyspread within boundaries 10 us and 1 eV, feasible for slow TOF1separation.

Continuous Ion Source

Referring to FIG. 7, the TOF-TOF method and apparatus of the inventionemploy a pulsed ion source (71), comprising a continuous ion source withsoft ionization (72) with an exit aperture (73), and a gas filled RFtrapping device (74), enclosed in an additional pumping stage (75).Continuous ion source is of the following list: electrospray (ESI),APCI, gas filled MALDI, PI or CI. The trapping device is of thefollowing list: 3-D Paul trap, linear RF only multipole with axialejection, curved RF multipole with radial ejection. The preference isgiven to linear quadrupole ion trap with axial ejection. The quadrupole(74) is surrounded by DC electrodes (76) and apertures (73, 77).

In operation, the quadrupole is filled with buffer gas at 1 to 100 mTorrpressure. Differential pumping system (75) reduces gas load on TOF1pumping. Ions are generated in the ion source (72) and continuously fillthe RF-only quadrupole ion guide (74). Gas collisions dampen ion kineticenergy and confine ions at quadrupole axis and at the bottom of DC wellcreated by electrodes (76) and aperture (77). Periodically, potential onelectrodes (76) and exit aperture (77) are adjusted to eject stored ionsin axial direction into TOF1. One can estimate that ion pulse has lessthan 1 eV energy spread and less than 10 us time spread.

In all above examples, pulsed ion sources are capable of generating ionpulse with less than 1 eV energy spread and less than 10 us time spread.A desired TOF1 mass resolution of 300 to 500, sufficient to separate agroup of isotopes, requires 600 to 1000 time resolution. Because of 10us initial time spread, the flight time for 1 kD ions has to be at least10 ms, achievable at few electron-Volts ion energy and effective flightpath from 5 to 10 m. The above described multi-turn TOF1 analyzersprovide 10 m effective path within a 0.5 to 1 m device. The next logicalquestion is whether ions could be fragmented within 10 us, so thatprimary separation would not be ruined.

Cid Cell

Referring to FIG. 8, the TOF-TOF method employs a short, high gaspressure CID cell (81) for ion fragmentation. The CID cell (81)comprises a vacuum housing (82), an entrance lens (83), a CID chamber(84) connected to gas inlet (85), an RF focusing device (86) withoptional DC electrodes (87), enclosed in the CID chamber, and exit ionlens (88). The CID cell also comprises an optional timed ion selectiongate (89). The gas inlet feeds buffer gas into CID chamber. The CIDchamber (83) comprises apertures (83A, B). The vacuum housing (82)comprises apertures (82A, B), and vacuum pump (82C). The RF focusingdevice is preferably a RF-only quadrupole.

Conventional CID cells, typically 10 to 20 cm long, operate at c.a 10mTorr gas pressure. In order to provide rapid ion transfer, the CIDcell, employed in the present invention, is much shorter, typically 5 to10 mm, and operates at much higher gas pressure, above 300 mTorr. Highpressure region is concentrated in the chamber 84 and is surrounded byadditional layer of differential pumping. Apertures 84A, B, typically1.5 mm diameter, limit total gas flow into vacuum housing to c.a 0.1Torr*L/s. Pump 82C with pumping speed of 300 L/s evacuates vacuumhousing to c.a 3 E-4 Torr. Apertures 82A, B, typically 1.5 mm diameter,further reduce gas flow into TOF1 and TOF2, operating at gas pressurebelow 3 E-7 Torr. To avoid gas discharge, the RF amplitude is reducedbelow 300V, accompanied by frequency drop below 1 MHz.

In operation, ions are accelerated in-front of the cell to energysufficient for ion fragmentation, typically 50 eV/kDa. Ion packets enterthe cell via apertures 82A and 84A, being focused by lens 83. At 300mTorr gas pressure, gas density equals n=1E+22m−3, and an ion of 1 kDmass with a cross section of □=100 A has a mean free path □=1/n□=0.1 mm.For typical quadrupole length of L=1 cm, ion experience c.a. 100collisions. Number of collisions, 3 times higher than ion/gas massratio, is sufficient to ensure fragmentation with subsequent dampening.First energetic collisions convert ion kinetic energy into ion heating,causing ion fragmentation. Once ions loose kinetic energy, subsequentgas collisions stabilize fragment ions, further dampens their kineticenergy and confine ions to axis due to RF field focusing. The phenomenonof collision dampening in CID cell is well described in U.S. Pat. No.______ by Don Douglas.

Time spread of ion beam in CID cell is of primary concern in the presentinvention. Travel time before high pressure region is assumed whiletuning TOF1, and it creates time delay only, not time spread. Gascollisions can cause significant time spread even in a short CID cell.To reduce the spread, ion passage through the cell is assisted byelectrostatic axial field, created by DC potentials of apertures 84A, B.At typical quadrupole inscribed diameter D=1 cm and length L=1 cm,fringing fields penetrate into RF quadrupole, being suppressed byfactor, less than 2. Accelerating potential of 20V can provide ion dragthrough gas at velocity c.a. 500 m/s, limiting full passage time below20 us and time spread below 10 us. Controlling passage time helps tobunch ions (i.e. compress duration of ion pulse) prior to injection intoTOF2. The accelerating field in CID cell is modulated, beingsynchronized (with time shift) to TOF2 injection pulses.

SID Cell

Referring to FIG. 9, the TOF-TOF method and apparatus of the inventionemploy a fragmentation cell (91) with surface induced dissociation (SID)for ion fragmentation. The SID cell (91) comprises a bunching (temporalfocusing), spatial focusing and steering lens (92), a probe (93), coatedwith fluorocarbon mono-layer, a pulse generator (94), attached to theprobe, a DC accelerating column (95), surrounded by ground shield (96).The DC accelerating column comprises a mesh (97), connected to a pulsegenerator (98).

In operation, ion packet of time separated parent ions is pulseaccelerated to c.a. 50 eV/kDa specific energy, being bunched by a lens(92). Bunching, previously employed in magnet sector-TOF tandems, isknown to compress ion packet duration below dT<1 us. The lens (92)focuses and steers parent ion packet (99) onto the center of the probe(93). Ion beam impinges the surface at some angle, say 45 degree. Mediumenergy collisions with fluorocarbon mono-layer surface are known toinduce fragmentation of peptides and small molecular ions. Fragment ionsbounce off the surface with c.a. 500 to 2000 m/s velocity, travellingless than 2 mm within dT<1 us of primary ion packet duration. Duringimpinging a small retarding potential is applied to the mesh 97,preventing leakage of fragment ions into the TOF2 analyzer. After anappropriate delay, corresponding to impinging of the entire primary ionpacket, pulse generators 94 and 98 are triggered, and electric pulsesare applied to the probe 93 and the mesh 97. Fragment ions are pulseaccelerated into the TOF2 analyzer.

Compared to the CID cell, the SID cell has advantages of:

-   -   1. operating at low pressure and thus reducing requirements on        pumping system    -   2. removing time spread in fragmentation step    -   3. accepting wider beam of primary ions.    -   4. Disadvantages of SID are    -   5. poorly characterized fragmentation pattern of medium mass        ions    -   6. higher energy spread of fragment ions, reducing TOF2        resolution    -   7. metastable decay of fragment ions in TOF2 analyzer.        The CID cell is better suited for in-line TOF1, while SID cell        is better suited for W-TOF1.

Referring to FIG. 10, the TOF-TOF method and apparatus of the inventionemploy a conventional orthogonal TOF (101) for mass analysis of fragmentions, preferably in conjunction with the CID cell. The o-TOF (101)comprises orthogonal pulse acceleration (102), an ion mirror (103), afloated free-flight region (104), a TOF detector (105) and an in-linedetector (106). Both detectors are connected to a data acquisitionsystem, comprising fast averaging transient recorder (107). TOF analyzer(101) is enclosed into vacuum chamber (108) and is evacuated by a pump(109).

Operation of o-TOF is well described in the art. Continuous or pulsedion beam, accelerated to c.a 10 eV, enters acceleration region. Periodicpulses accelerate ions orthogonal to c.a. 3 keV and inject ions into theTOF analyzer. Ions get reflected in the ion mirror and hit the TOFdetector 105. A portion of initial ion beam is acquired on the in-linedetector 106. To accommodate rapid analysis of fragment ions, parametersof the o-TOF are slightly altered. The analyzer is small—L=10 to 20 cm,operates at high TOF energy (5 to 15 kV) to accommodate high repetitionrate, c.a. 100 KHz. Small size analyzer allows operation at gas pressureslightly below 1 E-5 Torr. The conventional TOF analyzer is alsomodified by using high current secondary electron multiplier (SEM) orhybrid MCP/PEM for detector and by using a fast averaging transientrecorder for data acquisition system. Small length and short flight timepose limit on TOF2 resolution. To improve resolution of TOF2, one canincrease flight time in TOF2, while limiting time windows of admittedions by either of:

-   -   1. 10 us time gate interleaved between IMS scans and use slower        pulse rate of TOF2;    -   2. pulse TOF2 at 100 KHz rate and divert ions within TOF2 onto        several detectors;    -   3. pulse TOF2 at 100 KHz rate and use position sensitive        detector in TOF2.

TOF2 is optionally equipped with in-line detector in order to avoidacquiring signal in blank time, when no ions are coming from TOF1.

Conventional TOF2

Referring to FIG. 11, the TOF-TOF method also employs a conventionalreflecting TOF (111) for mass analysis of fragment ions, preferably inconjunction with the SID cell. The TOF (111) comprises a built-in SIDcell (91), an electrically floated free flight region (112), a detector(114) with a detector shield (113), an ion mirror (115), a vacuumhousing (116), a pump (117) and a transient recorder (118) for dataacquisition.

In operation, a pulse of fragment ions is accelerated within the SIDcell 91, fly through the field free region 112, get reflected in the ionmirror 115 and hit the detector 114. Ion trajectories are shown by lines119. Signal from the detector is acquired on the transient recorder 118.Again, for the purposes of rapid data acquisition the analyzer is short,L=10 to 20 cm, operates at high acceleration potential to accommodatehigh repetition rate of 100 KHz.

Having described individual components, it become easier to grasp theconcept and peculiarities of the integrated TOF-TOF method andapparatus. Below find specific examples of TOF-TOF tandems of theinvention, though, not limiting a multiplicity of viable combinations.

MS-MS with In-Line TOF-CID-o-TOF

Referring to FIG. 12, one preferred embodiment of TOF-TOF instrument(121) comprises sequentially connected pulsed source (71) withcontinuous ion source (72), the storage quadrupole (74) and electrodes(76,77), the in-line time-of-flight mass spectrometer TOF1 (31) with theRF-only quadrupole guide (32), surrounded by two pulsed ion mirrors(33A,B), the short gas-filled collision CID cell (81) with RF quadrupole(86), surrounded by apertures (84A,B) and the second, orthogonaltime-of-flight mass spectrometer o-TOF2 (101) with the pulse accelerator(102), equipped with analog data acquiring system (107). Individualcomponents have been described above and are shown on FIGS. 3, 7, 8 and10, and their previous numbers are retained in further discussion.

In operation, continuous ion source 71 feeds parent ions into thestorage quadrupole 74. Once in every 10 to 20 ms ions are ejected out ofstorage quadrupole, by pulsing potentials of DC electrodes 76 and ofexit aperture 77. Ejected ion packet, containing multiplicity of variousparent ions is less then 10 us long and has less than 1 eV energyspread. Mean energy of ejected ion pulse is adjusted to c.a. 2 eV byselecting pulse potentials of electrodes 76 and 77. Ions are admittedinto the TOF1 separator by dropping potential of the first mirror 33A.Ions are radial trapped by quadrupole RF field, but are free to travelalong the quadrupole axis. Once parent ions of all masses (limited tothe ratio Mmax/Mmin=2) pass the first mirror, the first mirror 33A isturned on. The second mirror 33B has been turned on within a previouscycle. Ions experience multiple reflections, preferably 5 reflections,between two mirrors with quadratic potential distribution along the TOF1axis. Period of oscillation is grossly independent on ion energy and isproportional to square root of parent ion mass. The effective flightpath of analyzer is up to 2□+1=7.3 times longer than the physical lengthof TOF1. After preferably 5 reflections ions are released out of theTOF1 by lowering-potential of the second mirror 33B. The train of timeseparated ion packets enters the CID cell. Typical time scale of timeseparation is of 10 ms, measured as a flight time of 1 kDa ions, andduration of each packet, corresponding to parent ion mass, isapproximately 10 us. Parent ions are separated with c.a 1000 timeresolution, corresponding to 500 mass resolution.

After leaving TOF1, each ion packet is accelerated to a specific energyof 50 eV/kDa, sufficient to induce fragmentation in gas collisions. Ionsare focused by lens system and injected into high pressure CID cell viaaperture 82A and 84A. Ions fragment in the cell, and fragment ions arecollision dampened and confined by RF field. The cell is activelyemptied by pulsed potential of two CID apertures 84A, B, synchronous andtime shifted relative to TOF2 pulses. Ions enter orthogonal accelerationregion 102, get injected into TOF2 analyzer, being time separated and,thus, mass analyzed in TOF2. Synchronized injection into TOF2 eliminatestime gaps, i.e. almost no fragments are lost between TOF2 pulses.Synchronous injection also improves duty cycle of TOF2. Most of fragmentions are contained within acceleration region 102 at the time of TOF2pulse.

TOF2 spectra present fragment spectra for every time separated parention mass. Spectra with the same TOF1 tag (i.e corresponding to parentions of the same m/z) are summed over multiple source injections. Within1 second acquisition the data will contain 1000 fragment spectra,averaged over 100 source injections.

In the above described apparatus there are three almost equal (c.a.10us) sources of time spread, deteriorating resolution of TOF1 separation:time spread gained in the ion source; time spread in the CID cell anddue to TOF2 digitization (i.e. acquiring spectra at discrete time).Assuming no correlation between those three sources, the overall timespread is estimated as 17 us (square root of three higher than eachspread). The resulting resolution of TOF1 separation becomes equal to300, which is still considered to be a fair resolution for parent ionseparation. For comparison, TOF1 resolution in commercial MALDI TOF-TOFis c.a 100, and quadrupole resolution in Q-TOF in a high sensitive modeis c.a 300. Resolution of TOF1 of the present invention can bepotentially improved by one of the following means:

-   -   Increasing length of TOF1 above 1 m;    -   Optimizing ion energy within TOF1;    -   Applying timed gate with multiple narrow mass windows,        interleaved between scans;    -   Pulsing TOF2 faster and diverting ions onto several detectors;    -   Using position sensitive detector in TOF2.

MS-MS with W-TOF-SID-coax TOF

Referring to FIG. 13, another preferred embodiment of TOF-TOF apparatusof the invention comprises the gas-filled pulsed MALDI ion source (61),the novel W-shape TOF1 (41), the SID cell (91) and the coaxial TOF2(111). The source 61 comprises, a gas-filled chamber (62), a sampleplate (63), a laser (65) and a low voltage bias supply (66), connectedto the sample plate 63. The TOF1 41 comprises deflection plates (45),two static reflectors (43) with terminating plates (44), and atwo-dimensional RF tunnel (42). Static reflectors (43) surround the RFchannel 42 to form a quadratic potential distribution. The SID cell 91comprises a bunching and focusing lens (92) and a probe (93), coatedwith fluorocarbon mono-layer. The TOF1 111 comprises a secondaryelectron multiplier-SEM (113), connected to a transient recorder (114).The source 61 and the SID cell 91 are located off-line to allow multipleion reflections within TOF1 41. The above selected combination ofelements is chosen mostly to demonstrate interaction between elements,not described in the previous TOF-TOF embodiment.

In operation, laser 65 pulses produce a short burst of primary ions offthe sample plate 63 at a repetition rate of 50 to 100 Hz. The sourcechamber 62 is filled with gas to relax ion internal energy and prevention decomposition. Ions are sampled through a thin gas layer by electricfield and gas flow, so that ion packet remains shorter than 10 us andhas energy spread less than 1 eV. Ion packet is accelerated by anotherfew Volts potential by low voltage bias supply 66 and get injected intothe multi-reflecting, TOF1 41 at a small angle to the Y axis. Thesteering plates 45 double the angle to reduce spatial spread in Xdirection, related to Y axis energy spread. Ion motion within TOF1 hasthree independent components—oscillation in confining RF field inZ-direction, multiple reflections along Y axis with period almostindependent on ion energy, and slow drift along orthogonal—X axis. Afterseveral Y bounces ions leave TOF1 and enter the bunching lens 92 of theSID cell 91, being time separated into a train of ion packets, alignedaccording to their m/z ratio. Multiple reflections at small ion energyallow prolonged time separation in the order of 10 ms. Since quadraticDC field in TOF1 compensates for ion energy spread, separation in TOF1does not increase the said 10 us time spread of ion packets. Thus, afterleaving TOF1 parent ions are separated with c.a. 300 to 500 massresolution.

Periodically, say once in every 10 us, ions are time bunched into c.a 1us packet and spatially focused to c.a 1 mm by a pulsed lens 92. Pulsefocused ion packets hit the surface of the SID probe 93, coated withfluorocarbon mono-layer. Collisions with surface induce ionfragmentation. Fragments, slowly moving from the surface, are spread forc.a 1 mm within 1 us time. A delayed electric pulse, applied to theprobe 93, accelerates fragment ions and injects them into the secondTOF2 111 analyzer. Initial parameters (i.e. parameters prior to theprobe pulse) of fragment ions are good enough to carry mass analysis inTOF2 with resolution of couple thousand. Signal is detected on the SEM114 with high dynamic range. Signal is passed to the transient recorder113, and data are acquired in a time-nested fashion. TOF2 transients,representing fragment spectra of various parent ions, are not mixedtogether. Each fragment mass spectrum obtains time tag of TOF1separation, measured as a time between source pulse and bunching lenspulse. TOF1 time tags carry information on parent ion m/z ratio. TOF2spectra with the same TOF1 time tag are averaged over multiple laserpulses to improve signal to noise ratio.

The inventor stresses the point that comprehensive TOF-TOF method of theinvention could be realized employing simpler static TOF1. Below findseveral examples of static separators. Retention of ion beam is staticfield requires operation at relatively higher energy around 100 eV.Millisecond separation time is achieved by extending flight path andusing focusing properties of specially designed electrostatic fields.

Referring to FIG. 14, another preferred embodiment of low energytime-of-flight separator (121) comprises an electrostatic lens (122), adeflector (123) and analyzer, consisting of entrance unit (124), twocoaxial electrodes (125) and (126) with DC voltage applied between them,and exit unit (127), followed by deflector (128) and lens (129). Thedescribed device is known as spiratron and is described in: Bakker I. M.B., The Spiratron. In: Adv. In Mass Spectrom., London, 1971, v.5, pp.278-280. The novelty is introduced by using the device as a low energyseparator in tandem TOF system.

In operation, ion beam from a pulsed ion source (71) is transformed bylens (122) into a much wider beam with proportionally lower angularspread (a “quasi-parallel beam”). This beam is deflected by deflector(123) to provide a controlled angle of inclination a to the axis ofelectrodes (125) and (126). It should be obvious to anybody skilled inthe art that the same effect could be achieved for example, bypositioning electrodes (125) and (126) at a fixed angle. The ion beamenters electrostatic radial field between electrodes (125) and (126) viaan aperture in the entrance unit (124). One preferred embodiment of theentrance unit (124) consists of 3 double-sided printed-circuit boards(PCB). Outside surfaces of these boards face deflector (123) and havemetallization on them to create an equipotential surface. Inner surfacesof these boards face the gap between electrodes (125) and (126) andcontain a set of metallization strips. These strips are connected to aresistive voltage divider that provides a voltage distribution matchingthe ideal logarithmic voltage distribution between electrodes (125) and(126) and thus minimizing perturbation of this field along iontrajectories. Exit unit (127) may have similar construction.

After ions pass through entrance unit (124), they start moving along aspiral trajectory, wound around electrode (125), and separate intime-of-flight according to their mass-to-charge ratios. To minimize ionbeam size, this spiral needs to be circular. This is achieved whenvoltage U between electrodes (125) and (126) corresponds to mean ionenergy V1 as ${U = {2V_{1}{\ln\left( \frac{r_{2}}{r_{1}} \right)}}},$where r1 and r2 are radii of electrodes (125) and (126) correspondingly.After a number of rotations, ions exit the field through the exit unit(127), after having drifted distance H along the axis. Construction ofthe exit unit (127) is similar to that of the injection unit (124). Themaximum number of rotations is limited mainly by full angular spread^(Δα) of the ion beam (^(Δα)<<1) that in its turn is limited byeffective temperature of the initial ion beam kT:${{\Delta\alpha} \approx {\frac{p}{M}\sqrt{\frac{kT}{V_{1}}}}},$where M is magnification of lens (122) and coefficient p depends on therequired confidence level (p≈4 for 95% of ions, p≈5 for 99% of ions, andp≈6.6 for 99.9% of ions). In the present example we choose M=5 and p=5,which will limit ^(Δα) to 1/45, i.e. approximately 1 degree. Then themaximum total length of trajectory is$L_{1} \approx \frac{H}{{\Delta\alpha} \cdot {\cos(\alpha)}} \approx {\frac{H \cdot M}{p}\sqrt{\frac{V_{1}}{kT}}}$

For example, for length H=0.5 m, kT=0.05 eV, V1=100 V, M=5, then totalflight path is L1∓22 m. Let us define ratio of time scales between TOF1and TOF2 as:${Ratio} = {{\frac{1}{2} \cdot \frac{TOF1}{TOF2}} = {{\frac{1}{2} \cdot \frac{L_{1}}{L_{2}}}\sqrt{\frac{V_{2}}{V_{1}}}}}$

This value defines the limit on the maximum mass resolving power of TOF1caused by the pulsed nature of TOF2. For the parameters above, effectivepath length of TOF2 L2=0.5 m and mean acceleration voltage V2=5000 V,Ratio≈150, which corresponds to mass resolution of TOF1 separation R˜75.Since resolution is also limited by relative energy spread of ion beamto c.a. R=100 it is not worth using longer device. Though resolution isinferior, compared to above described TOF1 spectrometers, the spiratrondevice has an advantage of simplicity, higher operation energy and itworks without stroboscopic techniques prior to TOF2. Resolution of 75 isstill useful in separating complex mixture of primary ions. Forcomparison separation in PSD MALDI has resolution from 50 to 100, andseparation in typical triple quadrupole experiments is typically around300.

Mean radius of the spiral r0 could be chosen on the basis of practicalconstraints, mainly the period d of metallization strips on boards124A-124C, For example, for r0=80 mm, step of the spiral is 15 mm. Ifd=3 mm, the resulting gap between the beam and plate (124C) ensuressufficient attenuation of fringing fields even for initial beam size 3-4mm after lens (122) (for M=5, this corresponds to ion beam diameter of0.6-0.8 mm on the exit from the source (71)).

The novel static low energy TOF can be coupled to any of above describedfragmentation means and TOF2 spectrometers or fragment analysis.Referring to FIG. 14 the TOF1 121 is coupled to the CID cell 81 and theorthogonal TOF 101. The major challenge in this combination is to focusthe primary beam onto the entrance of the CID cell. Though ion beam hashigh 100 eV energy and beam gets wider at the exit, the beam is grosslyparallel and can be well focused onto small aperture by conventionallens.

Multi-Pass TOF1

Referring to FIG. 15, another preferred embodiment of the first (i.e.TOF1) time of-flight separator of the invention (151), further called‘electrostatic multi-pass separator’, comprises a free flight channel(152), and two electrostatic mirrors (153), composed of focusingelectrodes (154), and reflector electrodes (155). The free flightchannel 152 has entrance and exit windows (156). All electrodes areextended along Y axis such that electrostatic field is two-dimensionalin the area of ion path. Pulsed ion beam is introduced into themulti-turn electrostatic TOF 151 via spatial focusing lens (157) and aset of steering plates (158). Ion path of ions is shown by the line(159). Typical axial potential distribution U(x) is shown by the graph160.

In operation, ion pulse is focused into a parallel beam by lens 158 andis steered by plates (159). The beam is introduced into the separator151 via the entrance window 156 at a small angle to X axis. Ionsexperience multiple reflections along X axis, while slowly driftingalong Y axis. After multiple full turns (each full turn is formed bypair of reflections) ions leave separator through the exit window 157,being time separated according to their m/z ratio. Number of full turnsdepends on injection angle—both adjustable by potentials on steeringplates.

Electrostatic mirrors are designed similar to mirror in griddles TOF,well known in the art. Electrostatic potentials, applied to mirrorelectrodes are tuned to satisfy conditions of spatial focusing andtime-of-flight focusing. Graph 160 shows the type of axial potentialdistribution U(x), satisfying those requirements. To provide spatialfocusing along Z direction, each of electrostatic mirrors 153 form alens with focal point, located near the center plane of free flightregion (shown by dashed line). Ion beam (line 159), starts as a parallelbeam at the entrance window 156. After first reflection in the rightside mirror the beam is focused into a point at the middle plane. Note,that focusing of all ions is presented on the drawing by single iontrajectory, intersecting the axis. After reflection in the left handmirror, the beam is again converted into a parallel beam.

According to inventor's ion optics simulation using SIMION program, thespatial focusing in the specific TOF1 151 is compatible withtime-of-flight focusing in at least first order, i.e. first derivativesof flight time on initial energy and on orthogonal displacement areequal to zero. Ion beam remains confined if only initial spatial spreadis under 5% of TOF1 width and angular spread is below 2 degrees. Forenergy spread under 3% the time of flight resolution of TOF1 exceeds10,000. Such initial conditions are realistic for ion beam acceleratedto approximately 30 electron Volts after pulse ejection out of linearstoring quadrupole.

Operation at relatively higher energy (30 to 100 eV), compared to otherembodiments, requires longer ion path in TOF1 (30 to 100 m) to achievemillisecond time scale separation in TOF1. Ion path could be easilyextended, because of low complexity of TOF1 design and its staticoperation. Instrument of 1 m long with approximately 20 full ion turnscorresponds to at least 50 m effective flight path.

Cylindrical Multi-Pass TOF1

Referring to FIG. 16, another preferred embodiment of the inventionpresents modified electrostatic multi-pass separator, formed by foldingtwo dimensional field into a cylindrical field. In this embodiment,called cylindrical multi-pass separator (161), for the purpose ofcompact design, each elongated electrode is converted into a pair ofcoaxial cylinders—internal and external. The separator 161 comprises afree flight channel, formed by cylinders (162,163), and twoelectrostatic mirrors, composed of focusing cylinders (164), andreflector cylinders (165). The external cylinder of free flight channel162 has entrance and exit windows (166), equipped with beam deflector(170). Pulsed ion beam, is introduced into separator 161 via spatialfocusing lens (167), via a set of steering plates (168), throughentrance window 166 and deflector 170. Ion path is shown by the line(169).

In operation, the cylindrical separator is very similar to the abovedescribed two-dimensional electrostatic multi-pass separator. Ions areforced to make multiple bounces between mirrors, while being spatiallyfocused by lens electrodes. In order to retain ions near same radius oforbit, an additional potential is applied between external and internalcylinders 162 and 163. Radial deflecting potential could be also appliedbetween external and internal cylinders of electrodes 164 and 165.

Entrance and exit of ions can be organized in multiple ways. FIG. 16Bshows an example of ion introduction through a slit-shaped window 166Bwith subsequent horizontal deflection, aligning ion beam along X-axis.To reduce fringing field, the deflector 170B is surrounded by mesh. FIG.16C shows an example of ion introduction along X axis through a segmentcut-out in the entire cylindrical analyzer. Beam is injected intoanalyzer after horizontal deflection by plates 170C. Field distortion isminimized by using double-sided PCB, equi-potential within cut-out andwith distributed potentials on the side oriented towards cylindricalanalyzer. The above described slow electrostatic multi-pass separatorsare suggested for use, in comprehensive tandem TOF spectrometer of theinvention in variety of combinations with earlier described pulsed ionsources, fragmentation cell and fast second TOF.

Obviously, presented examples of TOF1 separators, including separatorswith RF confinement, spiratron and static multi-pass separators, do notexhaust all the possibilities of TOF1, providing prolonged timeseparation, while retaining ion beam, but rather prove feasibility ofgeneral method of comprehensive tandem TOF mass spectrometry of theinvention.

Achieved Effect

The above described comprehensive tandem TOF spectrometers of theinvention are gaining speed and sensitivity of analysis, compared toexisting TOF-TOF mass spectrometers. The improvement is achieved byemploying principle of time-nested acquisition, first time applied totandem TOF. Ion pulses out of the ion source are fully utilized andmultiple parent ions are analyzed per single source pulse. The inventionalso improves the rate of MS-MS information, compared to closestprototype—IMS-TOF, also employing time-nested acquisition. Theimprovement is made by getting much higher resolution at the step ofparent ion separation and thus, providing analysis of more complexmixtures.

LC-LC-MS-MS

Much higher speed of tandem MS-MS analysis opens an opportunity ofcoupling multi-step liquid-phase separations with tandem MS analysis atrealistic time scale. Such separation techniques may include affinityseparation, liquid phase chromatography (LC) and capillaryelectrophoresis (CE). High speed LC and CE separation at few minutestime scale became routine in LC-MS analysis. However, LC-MS-MS analysisis usually slowed down by low speed of MS-MS stage, no longer the caseafter introducing the comprehensive TOF-TOF method and apparatus of theinvention.

Having described preferred embodiments and some examples of combininguseful elements; it will now become apparent for one skillful in the artthat other embodiments incorporating the concepts may be used. It isfelt, therefore, that these embodiments should not be limited todisclosed embodiments, but rather should be limited only by the spiritand the scope of the following claims. In claims, the ionmobility-orthogonal TOF is considered as the closest prototype.

1. A tandem mass spectrometer comprising sequentially connected pulsedion source, a parent ion separator, a fragmentation cell, a secondtime-of-flight mass spectrometer (TOF2) and a time nested dataacquisition system, acquiring fragment mass spectra for multiple parentions, wherein to improve resolution of parent ion separation the saidparent ion separator is a time-of-flight mass spectrometer and wherein,for ions of the same mass-to-charge ratio, time-of-flight in the saidTOF1 is significantly larger than both passage time through the saidfragmentation cell and time-of-light in the said TOF2.
 2. A tandem massspectrometer of claim 1, wherein time-of-flight in the said TOF1 is atleast 10 times larger than time-of-flight in the said TOF2.
 3. A tandemmass spectrometer of claim 1, wherein average ion energy in the saidTOF1 is at least 100 times smaller than in the said TOF2.
 4. The tandemmass spectrometer of claim 1, wherein the pulsed ion source is MALDI ionsource with gas pressure from vacuum up to 0.1 mbar.
 5. The tandem massspectrometer of claim 1, wherein the pulsed ion source comprises a pulseoperating radio frequency (RF) storage device and a continuous ionsource of the following list: an Electrospray source, a MALDI ionsource, filled with gas at gas pressure between 10 mtorr and 1 atm, anelectron impact ion source, electron impact with chemical ionization ionsource, or photo ionization ion source; ions are continuously suppliesfrom any of said ion sources to become accumulated and pulse-ejected outof the said storage device.
 6. The tandem mass spectrometer of claim 5,wherein said storage device comprises at least one RF-only linearmultipole, supplemented by at least one DC electrode, creating non-zeroaxial electric field.
 7. The tandem mass spectrometer of claim 1,wherein said TOF1 comprises RF only linear multipole, surrounded by twopulsed mirrors with axial quadratic electric field.
 8. The tandem massspectrometer of claim 1, wherein said TOF1 comprises two dimensionalRF-only ion tunnel, surrounded by two-dimensional DC mirrors withquadratic electric field.
 9. The tandem mass spectrometer of claim 1,wherein said TOF1 comprises at least a pair of coaxial electrodes withDC voltage applied between them and wherein ions are injected betweensaid electrodes at an angle to their axis.
 10. The tandem massspectrometer of claim 9, wherein ions enter and exit the gap betweensaid electrodes through a cut-off boundaries, which are formed by doublesided printed circuit boards
 11. The tandem mass spectrometer of claim1, wherein said TOF1 is a planar multi-pass electrostatic TOF,comprising a two-dimensional free flight channel and two planar focusingelectrostatic mirrors, composed of focusing and reflecting electrodes.12. The tandem mass spectrometer of claim 1, wherein said TOF1 is acylindrical multi-pass electrostatic TOF, comprising at least a pair ofcoaxial cylinders with radial deflection and two focusing electrostaticmirrors, composed of coaxial cylinders.
 13. The tandem mass spectrometerof claim 1, comprising an additional timed gate between said TOF1 andsaid fragmentation cell, which is capable of transmitting ions onlywithin multiple narrow time windows.
 14. The tandem mass spectrometer ofclaim 1, wherein energy of ion injection into said fragmentation cell isadjusted by the electrostatic offset between said TOF1 and saidfragmentation cell. The offset may be adjusted during TOF1 separation toprovide mass dependent ion energy around 50V/kD, optimum for parentfragmentation.
 15. The tandem mass spectrometer of claim 1, wherein saidfragmentation cell is collision induced dissociation (CID) cell, filledwith gas and comprising at least one RF-only multipole, supplemented byat least one DC electrode.
 16. The tandem mass spectrometer of claim 13,wherein the time spread of ion packet within said CID cell is reduced byusing short cell of less than 1 cm long, high gas pressure above 100mtorr.
 17. The tandem mass spectrometer of claim 13, wherein for thepurposes of time compression, the collision cell stores fragment ionsusing modulation of axial DC field within the cell, and ejects pulsedbeam synchronized with TOF2 pulses.
 18. The tandem mass spectrometer ofclaim 1, wherein said fragmentation cell comprises a pulsed temporal andspatial focusing lens and a target, coated by fluoro-hydrocarbonmonolayer.
 19. The tandem mass spectrometer of claim 1, wherein the TOF2is a TOF MS with an orthogonal time injection (o-TOF MS).
 20. The tandemmass spectrometer of claim 1, wherein said TOF2 comprises a high currentdetector and transient recorder.
 21. The tandem mass spectrometer ofclaim 1, wherein resolution of time separation in the TOF1 is enhancedby any of the following means: any reflector of the TOF1 forms quadraticpotential distribution along ion path, using large number of reflectionsin the TOF1, using TOF1 longer than 1 m, using said timed gate withmultiple narrow time windows.
 22. The tandem mass spectrometer of claim1, wherein an additional in-line detector is installed anywhere afterTOF1.
 23. Method of comprehensive MS-MS analysis, comprising thefollowing steps:
 24. pulse ejection of plurality of parent ions withvarious mass-to-charge ratio (M/Z) out of a pulsed ion source;
 25. timeseparation of parent ions within a first time separator; 26.fragmentation of time-separated ions;
 27. mass analysis of fragment ionswithin a second time-of-flight mass spectrometer (TOF2);
 28. Time nestedfragment mass spectra acquisition corresponding to multiple parent ionsper every single ion pulse without mixing fragment spectra of differentparent ions,
 29. wherein for the purpose of improving sensitivity andthroughput of MS-MS analysis, said time separation occurs within atime-of-flight mass spectrometer (TOF1) and wherein time of said parention separation significantly exceeds time of both said fragmentation andsaid fragment mass analysis.
 30. The method of comprehensive MS-MSanalysis, wherein said time of flight in the said TOF1 is at least 10times larger than in the said TOF2.
 31. The method of MS-MS analysis ofclaim 23, wherein the ion pulse is generated in a MALDI ion source withgas pressure from vacuum up to 100 mTorr.
 32. The method of MS-MSanalysis of claim 23, wherein said ion pulse is formed by pulsedejection out of storage quadrupole, while ions are introduced into thestorage quadrupole out a continuous ion source of the following list: anElectrospray source, a MALDI ion source, filled with gas at gas pressurebetween 10 mtorr and 1 atm, an electron impact ion source, electronimpact with chemical ionization ion source, or photo ionization ionsource.
 33. The method of MS-MS analysis of claim 23, wherein said timeseparation of parent ions occurs in quadratic DC field and whereinenergy of ions in said TOF1 is at least 100 times less than in saidTOF2.
 34. The method of MS-MS analysis of claim 26, wherein said timeseparation of parent ions in quadratic DC field is achieved withassistance of confining radio-frequency field in at least one dimension,orthogonal to DC field.
 35. The method of MS-MS analysis of claim 27,wherein said ion confinement in RF only field is achieved along oneaxis, ions are injected from one end of the RF field zone and aftermultiple reflections in pulsed quadratic DC field are released on theother end.
 36. The method of MS-MS analysis of claim 27, wherein theparent ion confinement by RF-only field is achieved along twodimensional plane. Ions are injected at small angle to TOF1 axis, whichis parallel to gradient of DC field. Ions experience multiplereflections in DC field, while slowly drifting in orthogonal direction,towards the exit of RF-filed.
 37. The method of MS-MS analysis of claim23, wherein said time separation of parent ions occurs in electrostaticfield and wherein the said energy of ions in the first time-of-flightseparator is at least 10 times smaller than in the said secondtime-of-flight mass spectrometer, and wherein the said effective flightpath in the said first time-of-flight separator is at least 30 timeslarger than in the said second time-of-flight mass spectrometer.
 38. Themethod of MS-MS analysis of claim 23, wherein said time separation ofparent ions occurs in electrostatic field, created by a pair of coaxialelectrodes, and wherein ions are injected into said electrostatic fieldat an angle to electrode axis, and wherein disturbance of saidelectrostatic field at boundaries is reduced by double sided printedcircuit boards.
 39. The method of MS-MS analysis of claim 23, whereinsaid time separation of parent ions occurs in planar electrostaticfield, formed by planar free flight channel and planar focusing griddlesion mirrors. Ions are injected at small angle to TOF1 axis andexperience multiple bounces between mirrors.
 40. The method of MS-MSanalysis of claim 23, wherein said time separation of parent ions occursin cylindrical electrostatic field, formed by multiple pairs of coaxialcylinders. In at least one pair radial field is applied. The cylindricalfield between cylinders is analogous to field of claim
 32. Ions areinjected at small angle to TOF1 axis and experience multiple bouncesbetween mirrors.
 41. The method of MS-MS analysis of claim 23, whereinresolution of time separation in TOF1 is enhanced by sampling multiplenarrow time widows before submitting ions to said fragmentation step.42. The method of MS-MS analysis of claim 23, where said fragmentationis achieved in one of the following processes: in energetic collisionswith gas, in collision with surface, by light.
 43. The method of MS-MSanalysis of claim 23, wherein the analysis is made in two steps: step 1of acquiring parent mass spectrum in TOF2, while using TOF1 in a passmode, and step 2 of sampling narrow time windows in front of collisioncell, corresponding to arrival of meaningful parent ions and acquiringfragment spectra for those time windows only. Said time windows areselected on the fly, based on parent masses out of stage 1 measurements.44. The method of MS-MS analysis of claim 23, wherein a “parent scan”,i.e. spectrum of parent ions having a predetermined set of fragmentions, is reconstructed out of a full MS/MS data set.
 45. Method ofLC-MS-MS analysis or LC-LC-MS-MS analysis, wherein the flow of solventis continuously introduced out of LC into a tandem mass spectrometer ofclaim 1 and MS-MS data are acquired using method, described in claims 21to 37.